Stabilized fibronectin domain compositions, methods and uses

ABSTRACT

A protein scaffold based on a consensus sequence of fibronectin type III (FN3) proteins, such as the tenth FN3 repeat from human fibronectin (human Tenascin), including isolated nucleic acids that encode a protein scaffold, vectors, host cells, and methods of making and using thereof, exhibit enhanced thermal and chemical stability while presenting six modifiable loop domains which can be engineered to form a binding partner capable of binding to a target for applications in diagnostic and/or therapeutic compositions, methods and devices.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims priority to U.S. Provisional Application Ser.No. 61/329,980, filed 30 Apr. 2010, the entire contents of which isincorporated herein by reference in their entirety.

BACKGROUND

1. Field of the Invention

The present invention relates to protein scaffolds with novelproperties, including the ability to bind to cellular targets. Moreparticularly, the present invention is directed to a protein scaffoldbased on a consensus sequence of a fibronectin type III (FN3) repeat.

2. Discussion of the Field

Monoclonal antibodies are the most widely used class of therapeuticproteins when high affinity and specificity for a target molecule aredesired. However, non-antibody proteins that can be engineered to bindsuch targets are also of high interest in the biopharmaceuticalindustry. These “alternative scaffold” proteins may have advantages overtraditional antibodies due to their small size, lack of disulphidebonds, high stability, and ability to be expressed in prokaryotic hosts.Novel methods of purification are readily applied; they are easilyconjugated to drugs/toxins, penetrate efficiently into tissues and arereadily formatted into multispecific binders (Skerra 2000 J Mol Recognit13(4): 167-87; Binz and Pluckthun 2005 Curr Opin Biotechnol 16(4):459-69).

One such alternative scaffold is the immunoglobulin (Ig) fold. This foldis found in the variable regions of antibodies, as well as thousands ofnon-antibody proteins. It has been shown that one such Ig protein, thetenth fibronectin type III (FN3) repeat from human fibronectin, cantolerate a number of mutations in surface exposed loops while retainingthe overall Ig-fold structure. Thus, libraries of amino acid variantshave been built into these loops and specific binders selected to anumber of different targets (Koide et al. 1998 J Mol Biol 284(4):1141-51; Karatan et al. 2004 Chem Biol 11(6): 835-44). Such engineeredFN3 domains have been found to bind to targets with high affinity, whileretaining important biophysical properties (Parker et al. 2005 ProteinEng Des Sel 18(9): 435-44).

Desirable physical properties of potential alternative scaffoldmolecules include high thermal stability and reversibility of thermalfolding and unfolding. Several methods have been applied to increase theapparent thermal stability of proteins and enzymes, including rationaldesign based on comparison to highly similar thermostable sequences,design of stabilizing disulfide bridges, mutations to increasealpha-helix propensity, engineering of salt bridges, alteration of thesurface charge of the protein, directed evolution, and composition ofconsensus sequences (Lehmann and Wyss 2001 Curr Opin Biotechnol 12(4):371-5). High thermal stability is a desired property of such scaffoldsas it may increase the yield of recombinant protein obtained, improvesolubility of the purified molecule, improve activity of intracellularscaffolds, decrease immunogenicity, and minimize the need of a coldchain in manufacturing.

SUMMARY OF THE INVENTION

The present invention provides a protein scaffold based on a fibronectintype III (FN3) repeat protein, encoding or complementary nucleic acids,vectors, host cells, compositions, combinations, formulations, devices,and methods of making and using them. In a preferred embodiment, theprotein scaffold is comprised of a consensus sequence of multiple FN3domains from human Tenascin-C (hereinafter “Tenascin”). In a furtherpreferred embodiment, the protein scaffold of the present invention is aconsensus sequence of 15 FN3 domains (SEQ ID NO: 1-15) or a variantthereof. In a particular aspect of the invention, the protein scaffoldof the invention has substitute residues which cause the scaffoldprotein to demonstrate enhanced ability to resist thermal and chemicaldenaturation. The protein scaffolds of the invention can be engineeredby methods known in the art, including inserting residues at designatedloop regions within the scaffold, to form a binding domain selective fora binding partner. The binding partner may be a soluble molecule or acellularly anchored molecule, for example, the extracellular domain of areceptor protein.

In one embodiment, specific substitutions of the in the consensus-basedsequence of SEQ ID NO: 16 (Tencon) selected for inherent thermal andchemical stability described herein improve the thermal stability of theTencon scaffold by up to 11° C. and shift the mid-point of GdmCl induceddenaturation from 3.4 M to greater than 5 M. In one embodiment, thespecific substitutions to SEQ ID NO: 16 (Tencon) are unitary, such asN46V, E14P, and E86I, and, in an alternative embodiment thesubstitutions are multiple, such as N46V and E86I, all of E14P and N46Vand E86I, and all of L17A and N46V and E86I. Tencon-based polypeptideswith enhanced stability provide scaffolds with improved ease ofpurification, formulation, and increased shelf-life. Engineered bindingpartners with improved overall stability can be produced by introducingrandomized peptides into loops of the stabilized scaffold.

The protein scaffolds of the invention may be used as monomeric units orlinked to form polymeric structures with the same or different bindingpartner specificity. The Tencon protein scaffold-based molecules may befurther modified to enhance one or more in vivo properties related tobiodistribution, persistence in the body, or therapeutic efficacy suchas the association with molecules which alter cellular, particularly,epithelial cell uptake, for example, the Fc region of an antibody, ormolecules designed to bind serum proteins such as an albumin bindingdomain. In further embodiments, the protein scaffolds of the inventionmay be bound to a nucleic acid molecule that may encode the proteinscaffold.

The present invention also provides at least one method for expressingat least one protein scaffold polypeptide whose sequence is related to aconsensus sequence of multiple FN3 domains, in a host cell, comprisingculturing a host cell as described herein under conditions wherein atleast one protein scaffold is expressed in detectable and/or recoverableamounts.

The present invention also provides at least one composition comprising(a) a protein scaffold based on a consensus sequence of multiple FN3domains and/or encoding nucleic acid as described herein; and (b) asuitable and/or pharmaceutically acceptable carrier or diluent.

The present invention further comprises a method of generating librariesof a protein scaffold based on a fibronectin type III (FN3) repeatprotein, preferably, a consensus sequence of multiple FN3 domains and,more preferably, a consensus sequence of multiple FN3 domains from humanTenascin with enhanced thermal and chemical stability. Libraries can begenerated by altering the amino acid composition of a single loop or thesimultaneous alteration of multiple loops or additional positions of thescaffold molecule. The loops that are altered can be lengthened orshortened accordingly. Such libraries can be generated to include allpossible amino acids at each position, or a designed subset of aminoacids. The library members can be used for screening by display, such asin vitro display (DNA, RNA, ribosome display, etc.), yeast, bacterial,and phage display.

The protein scaffolds of the present invention provide enhancedbiophysical properties, such as stability under conditions of highosmotic strength and solubility at high concentrations. The domains ofthe scaffold proteins are not disulfide bonded, making them capable ofexpression and folding in systems devoid of enzymes required fordisulfide linkage formation, including prokaryotic systems, such as E.coli, and in in vitro transcription/translation systems, such as therabbit reticulocyte lysate system.

In an additional aspect, the present invention provides a method ofgenerating a scaffold molecule that binds to a particular target bypanning the scaffold library of the invention with the target anddetecting binders. In other related aspects, the invention comprisesscreening methods that may be used to generate or affinity matureprotein scaffolds with the desired activity, e.g., capable of binding totarget proteins with a certain affinity. Affinity maturation can beaccomplished by iterative rounds of mutagenesis and selection usingsystems, such as phage display or in vitro display. Mutagenesis duringthis process may be the result of site directed mutagenesis to specificscaffold residues, random mutagenesis due to error-prone PCR, DNAshuffling, and/or a combination of these techniques. The presentinvention further provides any invention described herein.

BRIEF DESCRIPTION OF THE DRAWING

FIG. 1. SDS-PAGE analysis of purified Tencon performed on a NuPAGE 4-12%Bis-Tris gel (Invitrogen) and stained with coomassie blue. N stands fornative conditions and R for reduced conditions.

FIG. 2 shows a circular dichroism analysis of Tencon in PBS.

FIG. 3 shows a circular dichroism analysis of the third FN3 domain fromtenascin and Tencon in PBS where the melting temperatures of 54° C. and78° C. were obtained respectively.

FIG. 4 shows phagemid plasmid design of pTencon-pIX. Expression isdriven by a Lac promoter and secretion via the OmpA signal sequence.

FIG. 5 shows myc-Tencon can be displayed on M13 phage using ELISAdemonstrating the binding of phage to anti-Myc coated, CNTO95 coated,and uncoated wells.

FIG. 6 is a drawing depicting the loop structure of the third FN3 domainof human Tenascin.

FIG. 7 shows the screening by ELISA output of IgG selections wherebyindividual clones were tested for binding to biotinylated IgG orbiotinylated HSA as a control.

FIGS. 8A-B are graphs showing the GdmCl induced denaturation for singlemutants (A) and combinatorial mutants (B) as measured by fluorescenceexcitation of 280 nm and an emission of 360 nm.

DETAILED DESCRIPTION OF THE INVENTION Abbreviations

ADCC=antibody-dependent cellular cytotoxicity; CDC=complement-dependentcytotoxicity; DSC=differential scanning calorimetry; ΔG=Gibbs FreeEnergy; IgG=immunoglobulin G; Tm=temperature of melting;

Definitions & Explanation of Terminology

The term “antibody” or “antibody moiety” is intended to encompassantibodies, digestion fragments, specified portions and variantsthereof, including, without limitation, antibody mimetics or comprisingportions of antibodies that mimic the structure and/or function of anantibody or specified fragment or portion thereof, including, withoutlimitation, single chain antibodies, single domain antibodies,minibodies, and fragments thereof. Functional fragments includeantigen-binding fragments that bind to the target antigen of interest.For example, antibody fragments capable of binding to a target antigenor portions thereof, including, but not limited to, Fab (e.g., by papaindigestion), Fab′ (e.g., by pepsin digestion and partial reduction) andF(ab′)₂ (e.g., by pepsin digestion), facb (e.g., by plasmin digestion),pFc′ (e.g., by pepsin or plasmin digestion), Fd (e.g., by pepsindigestion, partial reduction and reaggregation), Fv or scFv (e.g., bymolecular biology techniques) fragments, are encompassed by the termantibody. The antibody or fragment may be derived from any mammal, suchas, but not limited to, a human, a mouse, a rabbit, a rat, a rodent, aprimate, a camelid, a goat, or any combination thereof and includesisolated human, primate, rodent, mammalian, chimeric, humanized and/orCDR-grafted antibodies, immunoglobulins, cleavage products and otherspecified portions and variants thereof.

The term “epitope” means a protein determinant capable of specificbinding to an antibody or engineered binding domain such as one or moreloops of a scaffold-based protein. Epitopes usually consist ofchemically active surface groupings of molecules such as amino acids orsugar side chains and usually have specific three-dimensional structuralcharacteristics, as well as specific charge characteristics.Conformational and nonconformational epitopes are distinguished in thatthe binding to the former but not the latter is lost in the presence ofdenaturing solvents. The conformational epitopes result fromconformational folding of the target molecule which arise when aminoacids from differing portions of the linear sequence of the targetmolecule come together in close proximity in 3-dimensional space. Suchconformational epitopes are typically distributed on the extracellularside of the plasma membrane.

The terms “Fc,” “Fc-containing protein” or “Fc-containing molecule” asused herein refer to a monomeric, dimeric or heterodimeric proteinhaving at least an immunoglobulin CH2 and CH3 domain. The CH2 and CH3domains can form at least a part of the dimeric region of theprotein/molecule (e.g., antibody).

The term “stability” as used herein refers to the ability of a moleculeto maintain a folded state under physiological conditions such that itretains at least one of its normal functional activities, for example,binding to a target molecule like a cytokine or serum protein.Measurement of protein stability and protein liability can be viewed asthe same or different aspects of protein integrity. Proteins aresensitive or “labile” to denaturation caused by heat, by ultraviolet orionizing radiation, changes in the ambient osmolarity and pH if inliquid solution, mechanical shear force imposed by small pore-sizefiltration, ultraviolet radiation, ionizing radiation, such as by gammairradiation, chemical or heat dehydration, or any other action or forcethat may cause protein structure disruption. The stability of themolecule can be determined using standard methods. For example, thestability of a molecule can be determined by measuring the thermal melt(“TM”) temperature. The TM is the temperature in ° Celsius (° C.) atwhich ½ of the molecules become unfolded. Typically, the higher the TM,the more stable the molecule. In addition to heat, the chemicalenvironment also changes the ability of the protein to maintain aparticular three dimensional structure.

Chemical denaturation can likewise be measured by a variety of methods.A chemical denaturant is an agent known to disrupt non-covalentinteractions and covalent bonds within a protein, including hydrogenbonds, electrostatic bonds, Van der Waals forces, hydrophobicinteractions, or disulfide bonds. Chemical denaturants includeguanidinium hydrochloride, guanadinium thiocyanate, urea, acetone,organic solvents (DMF, benzene, acetonitrile), salts (ammonium sulfatelithium bromide, lithium chloride, sodium bromide, calcium chloride,sodium chloride); reducing agents (e.g. dithiothreitol,beta-mercaptoethanol, dinitrothiobenzene, and hydrides, such as sodiumborohydride), non-ionic and ionic detergents, acids (e.g. hydrochloricacid (HCl), acetic acid (CH₃COOH), halogenated acetic acids),hydrophobic molecules (e.g. phospholipids), and targeted denaturants(Jain R. K and Hamilton A. D., Angew. Chem. 114(4), 2002). Quantitationof the extent of denaturation can rely on loss of a functional propertysuch as ability to bind a target molecule, or by physiochemicalproperties such tendency to aggregation, exposure of formerly solventinaccessible residues, or disruption or formation of disulfide bonds.

In terms of loss of stability, i.e. “denaturing” or “denaturation” of aprotein is meant the process where some or all of the three-dimensionalconformation imparting the functional properties of the protein has beenlost with an attendant loss of activity and/or solubility. Forcesdisrupted during denaturation include intramolecular bonds, includingbut not limited to electrostatic, hydrophobic, Van der Waals forces,hydrogen bonds, and disulfides. Protein denaturation can be caused byforces applied to the protein or a solution comprising the protein suchas mechanical force (for example, compressive or shear-force), thermal,osmotic stress, change in pH, electrical or magnetic fields, ionizingradiation, ultraviolet radiation and dehydration, and by chemicaldenaturants.

A “therapeutically effective” treatment or amount as used herein, refersto an amount of sufficient quantity to cause a detectable lessening oramelioration of the cause of a disorder or its symptoms. “Ameliorate”refers to a lessening of the detrimental effect of the disorder in thepatient receiving the therapy. The subject of the invention ispreferably a human, however, it can be envisioned that any animal inneed of a treatment for a deleterious conditions, disorder, or diseasecan be treated with a scaffold-based protein designed for that purpose.

Overview

The present invention provides an isolated, recombinant and/or syntheticprotein scaffold based on a consensus sequence of a fibronectin type III(FN3) repeat protein, including, without limitation, mammalian-derivedscaffold, as well as compositions and encoding nucleic acid moleculescomprising at least one polynucleotide encoding a protein scaffold basedon the consensus FN3 sequence. The present invention further includes,but is not limited to, methods of making and using such nucleic acidsand protein scaffolds, including as a discovery platform, and fordiagnostic and therapeutic compositions, methods and devices.

The protein scaffolds of the present invention offer advantages overlarger immunoglobulin based biotherapeutics, owing to their small,compact size. In particular, the size and shape of a biologic moleculecan impact its ability to be administered locally, orally, or cross theblood-brain barrier; ability to be expressed in low cost systems such asE. coli; ability to be engineered into bi- or multi-specific moleculesbinding to multiple targets or multiple epitopes of the same target,suitability for conjugation, i.e. to actives, polymers, and probes;ability to be formulated to high concentrations; and the ability of suchmolecules to effectively penetrate diseased tissues and tumors.

Moreover, the protein scaffolds possess many of the properties ofantibodies in relation to their fold that mimics the variable region ofan antibody. This orientation enables the FN3 loops to be exposedsimilar to antibody complementarity determining regions (CDRs). Theyshould be able to bind to cellular targets and the loops can be altered,e.g., affinity matured, to improve certain binding or relatedproperties.

Three of the six loops of the protein scaffold of the inventioncorrespond topologically to the binding domains of an antibodypositioned at the loops of the variable domain known to be hypervariablein nature (the hypervariable domains loops (HVL), at positions asdefined by Kabat as the residues of the complementarity determiningregions (CDRs), i.e., antigen-binding regions, of an antibody, while theremaining three loops are surface exposed in a manner similar toantibody CDRs. These loops span or are positioned at or about residues13-16, 22-28, 38-43, 51-54, 60-64, and 75-81 of SEQ ID NO:16 as shown inTable 3 below and FIG. 6. Preferably, the loop regions at or aboutresidues 22-28, 51-54, and 75-81 are altered for binding specificity andaffinity. One or more of these loop regions are randomized with otherloop regions and/or other strands maintaining their sequence as backboneportions to populate a library and potent binders can be selected fromthe library having high affinity for a particular protein target. One ormore of the loop regions can interact with a target protein similar toan antibody CDR interaction with the protein.

The scaffolds of the present invention may incorporate other subunits,e.g., via covalent interaction. All or a portion of an antibody constantregion may be attached to the scaffold to impart antibody-likeproperties especially those properties associated with the Fc region,e.g., complement activity (ADCC), half-life, etc. For example, effectorfunction can be provided and/or controlled, e.g., by modifying C1qbinding and/or FcγR binding and thereby changing CDC activity and/orADCC activity. “Effector functions” are responsible for activating ordiminishing a biological activity (e.g., in a subject). Examples ofeffector functions include, but are not limited to: C1q binding;complement dependent cytotoxicity (CDC); Fc receptor binding;antibody-dependent cell-mediated cytotoxicity (ADCC); phagocytosis; downregulation of cell surface receptors (e.g., B cell receptor; BCR), etc.Such effector functions may require the Fc region to be combined with abinding domain (e.g., protein scaffold loops) and can be assessed usingvarious assays (e.g., Fc binding assays, ADCC assays, CDC assays, etc.).

Additional moieties may be appended or associated with thescaffold-based polypeptide or variant such as a toxin conjugate, albuminor albumin binders, polyethylene glycol (PEG) molecules may be attachedto the scaffold molecule for desired properties. These moieties may bein-line fusions with the scaffold coding sequence and may be generatedby standard techniques, for example, by expression of the fusion proteinfrom a recombinant fusion encoding vector constructed using publiclyavailable coding nucleotide sequences. Alternatively, chemical methodsmay be used to attach the moieties to a recombinantly producedscaffold-based protein.

The scaffolds of the present invention can be used as monospecific inmonomeric form or as bi- or multi-specific (for different proteintargets or epitopes on the same protein target) in multimer form. Theattachments between each scaffold unit may be covalent or non-covalent.For example, a dimeric bispecific scaffold has one subunit withspecificity for a first target protein or epitope and a second subunitwith specificity for a second target protein or epitope. Scaffoldsubunits can be joined in a variety of conformations that can increasethe valency and thus the avidity of antigen binding.

Generation and Production of Scaffold Protein

At least one scaffold protein of the present invention can be optionallyproduced by a cell line, a mixed cell line, an immortalized cell orclonal population of immortalized cells, as well known in the art. See,e.g., Ausubel, et al., ed., Current Protocols in Molecular Biology, JohnWiley & Sons, Inc., NY, N.Y. (1987-2001); Sambrook, et al., MolecularCloning: A Laboratory Manual, 2^(nd) Edition, Cold Spring Harbor, N.Y.(1989); Harlow and Lane, Antibodies, a Laboratory Manual, Cold SpringHarbor, N.Y. (1989); Colligan, et al., eds., Current Protocols inImmunology, John Wiley & Sons, Inc., NY (1994-2001); Colligan et al.,Current Protocols in Protein Science, John Wiley & Sons, NY, N.Y.,(1997-2001).

Amino acids from a scaffold protein can be altered, added and/or deletedto reduce immunogenicity or reduce, enhance or modify binding, affinity,on-rate, off-rate, avidity, specificity, half-life, stability,solubility or any other suitable characteristic, as known in the art.

Bioactive scaffold-based proteins can be engineered with retention ofhigh affinity for the antigen and other favorable biological properties.To achieve this goal, the scaffold proteins can be optionally preparedby a process of analysis of the parental sequences and variousconceptual engineered products using three-dimensional models of theparental and engineered sequences. Three-dimensional models are commonlyavailable and are familiar to those skilled in the art. Computerprograms are available which illustrate and display probablethree-dimensional conformational structures of selected candidatesequences and can measure possible immunogenicity (e.g., Immunofilterprogram of Xencor, Inc. of Monrovia, Calif.). Inspection of thesedisplays permits analysis of the likely role of the residues in thefunctioning of the candidate sequence, i.e., the analysis of residuesthat influence the ability of the candidate scaffold protein to bind itsantigen. In this way, residues can be selected and combined from theparent and reference sequences so that the desired characteristic, suchas affinity for the target antigen(s), is achieved. Alternatively, or inaddition to, the above procedures, other suitable methods of engineeringcan be used.

Screening

Screening engineered scaffold-based protein or libraries comprisingscaffold-based proteins with variegated residues or domains for specificbinding to similar proteins or fragments can be conveniently achievedusing nucleotide (DNA or RNA display) or peptide display libraries, forexample, in vitro display. This method involves the screening of largecollections of peptides for individual members having the desiredfunction or structure. The displayed peptide with or without nucleotidesequences can be from 3 to 5000 or more nucleotides or amino acids inlength, frequently from 5-100 amino acids long, and often from about 8to 25 amino acids long. In addition to direct chemical synthetic methodsfor generating peptide libraries, several recombinant DNA methods havebeen described. One type involves the display of a peptide sequence onthe surface of a bacteriophage or cell. Each bacteriophage or cellcontains the nucleotide sequence encoding the particular displayedpeptide sequence.

The protein scaffolds of the invention can bind human or other mammalianproteins with a wide range of affinities (K_(D)). In a preferredembodiment, at least one protein scaffold of the present invention canoptionally bind to a target protein with high affinity, for example,with a K_(D) equal to or less than about 10⁻⁷ M, such as but not limitedto, 0.1-9.9 (or any range or value therein)×10⁻⁸, 10⁻⁹, 10⁻¹⁰, 10⁻¹¹,10⁻¹², 10⁻¹³, 10⁻¹⁴, 10⁻¹⁵ or any range or value therein, as determinedby surface plasmon resonance or the Kinexa method, as practiced by thoseof skill in the art.

The affinity or avidity of a protein scaffold for an antigen can bedetermined experimentally using any suitable method. (See, for example,Berzofsky, et al., “Antibody-Antigen Interactions,” In FundamentalImmunology, Paul, W. E., Ed., Raven Press: New York, N.Y. (1984); Kuby,Janis Immunology, W. H. Freeman and Company: New York, N.Y. (1992); andmethods described herein). The measured affinity of a particular proteinscaffold-antigen interaction can vary if measured under differentconditions (e.g., osmolarity, pH). Thus, measurements of affinity andother antigen-binding parameters (e.g., K_(D), K_(on), K_(off)) arepreferably made with standardized solutions of protein scaffold andantigen, and a standardized buffer, such as the buffer described herein.

Competitive assays can be performed with the protein scaffold of thepresent invention in order to determine what proteins, antibodies, andother antagonists compete for binding to a target protein with theprotein scaffold of the present invention and/or share the epitoperegion. These assays as readily known to those of ordinary skill in theart evaluate competition between antagonists or ligands for a limitednumber of binding sites on a protein. The protein and/or antibody isimmobilized, isolated, or captured before or after the competition andthe sample bound to the target protein is separated from the unboundsample, for example, by decanting (where the protein/antibody waspreinsolubilized) or by centrifuging (where the protein/antibody wasprecipitated after the competitive reaction). Also, the competitivebinding may be determined by whether function is altered by the bindingor lack of binding of the protein scaffold to the target protein, e.g.,whether the protein scaffold molecule inhibits or potentiates theenzymatic activity of, for example, a label. ELISA and other functionalassays may be used, as well known in the art.

Nucleic Acid Molecules

Nucleic acid molecules of the present invention encoding proteinscaffolds can be in the form of RNA, such as mRNA, hnRNA, tRNA or anyother form, or in the form of DNA, including, but not limited to, cDNAand genomic DNA obtained by cloning or produced synthetically, or anycombinations thereof. The DNA can be triple-stranded, double-stranded orsingle-stranded, or any combination thereof. Any portion of at least onestrand of the DNA or RNA can be the coding strand, also known as thesense strand, or it can be the non-coding strand, also referred to asthe anti-sense strand.

Isolated nucleic acid molecules of the present invention can includenucleic acid molecules comprising an open reading frame (ORF),optionally, with one or more introns, e.g., but not limited to, at leastone specified portion of at least one protein scaffold; nucleic acidmolecules comprising the coding sequence for a protein scaffold or loopregion that binds to the target protein; and nucleic acid moleculeswhich comprise a nucleotide sequence substantially different from thosedescribed above but which, due to the degeneracy of the genetic code,still encode the protein scaffold as described herein and/or as known inthe art. Of course, the genetic code is well known in the art. Thus, itwould be routine for one skilled in the art to generate such degeneratenucleic acid variants that code for specific protein scaffolds of thepresent invention. See, e.g., Ausubel, et al., supra, and such nucleicacid variants are included in the present invention.

As indicated herein, nucleic acid molecules of the present inventionwhich comprise a nucleic acid encoding a protein scaffold can include,but are not limited to, those encoding the amino acid sequence of aprotein scaffold fragment, by itself; the coding sequence for the entireprotein scaffold or a portion thereof; the coding sequence for a proteinscaffold, fragment or portion, as well as additional sequences, such asthe coding sequence of at least one signal leader or fusion peptide,with or without the aforementioned additional coding sequences, such asat least one intron, together with additional, non-coding sequences,including but not limited to, non-coding 5′ and 3′ sequences, such asthe transcribed, non-translated sequences that play a role intranscription, mRNA processing, including splicing and polyadenylationsignals (for example, ribosome binding and stability of mRNA); anadditional coding sequence that codes for additional amino acids, suchas those that provide additional functionalities. Thus, the sequenceencoding a protein scaffold can be fused to a marker sequence, such as asequence encoding a peptide that facilitates purification of the fusedprotein scaffold comprising a protein scaffold fragment or portion.

Nucleic Acid Molecules

The invention also provides for nucleic acids encoding the compositionsof the invention as isolated polynucleotides or as portions ofexpression vectors including vectors compatible with prokaryotic,eukaryotic or filamentous phage expression, secretion and/or display ofthe compositions or directed mutagens thereof.

The isolated nucleic acids of the present invention can be made using(a) recombinant methods, (b) synthetic techniques, (c) purificationtechniques, and/or (d) combinations thereof, as well-known in the art.

The polynucleotides useful in the practice of the present invention willencode a functional portion of the a protein scaffold described herein.The polynucleotides of this invention embrace nucleic acid sequencesthat can be employed for selective hybridization to a polynucleotideencoding a protein scaffold of the present invention. The presentinvention provides isolated nucleic acids that hybridize under selectivehybridization conditions to a polynucleotide disclosed herein. Thus, thepolynucleotides of this embodiment can be used for isolating, detecting,and/or quantifying nucleic acids comprising such polynucleotides. Forexample, polynucleotides of the present invention can be used toidentify, isolate, or amplify partial or full-length clones in adeposited library. In some embodiments, the polynucleotides are genomicor cDNA sequences isolated, or otherwise complementary to, a cDNA from ahuman or mammalian nucleic acid library.

The nucleic acids can conveniently comprise sequences in addition to apolynucleotide of the present invention. For example, a multi-cloningsite comprising one or more endonuclease restriction sites can beinserted into the nucleic acid to aid in isolation of thepolynucleotide. Also, translatable sequences can be inserted to aid inthe isolation of the translated polynucleotide of the present invention.For example, a hexa-histidine marker sequence provides a convenientmeans to purify the proteins of the present invention. The nucleic acidof the present invention, excluding the coding sequence, is optionally avector, adapter, or linker for cloning and/or expression of apolynucleotide of the present invention.

Additional sequences can be added to such cloning and/or expressionsequences to optimize their function in cloning and/or expression, toaid in isolation of the polynucleotide, or to improve the introductionof the polynucleotide into a cell. Use of cloning vectors, expressionvectors, adapters, and linkers is well known in the art.

As indicated herein, nucleic acid molecules of the present inventionwhich comprise a nucleic acid encoding a protein scaffold can include,but are not limited to, those encoding the amino acid sequence of aprotein scaffold fragment, by itself; the coding sequence for the entireprotein scaffold or a portion thereof; the coding sequence for a proteinscaffold, fragment or portion, as well as additional sequences, such asthe coding sequence of at least one signal leader or fusion peptide,with or without the aforementioned additional coding sequences, such asat least one intron, together with additional, non-coding sequences,including but not limited to, non-coding 5′ and 3′ sequences, such asthe transcribed, non-translated sequences that play a role intranscription, mRNA processing, including splicing and polyadenylationsignals (for example, ribosome binding and stability of mRNA); anadditional coding sequence that codes for additional amino acids, suchas those that provide additional functionalities. Thus, the sequenceencoding a protein scaffold can be fused to a marker sequence, such as asequence encoding a peptide that facilitates purification of the fusedprotein scaffold comprising a protein scaffold fragment or portion.

For bacterial expression including phage infected bacteria, a preferredsecretion signal is a pelB or ompA secretion signal but other secretionsignal polypeptide domains may be used as described in U.S. Pat. No.5,658,727. In phage display, a downstream translatable DNA sequenceencodes a filamentous phage coat protein, e.g. pIII or pIX protein.Preferred phage proteins are obtainable from filamentous phage M13, fl,fd, and the like equivalent filamentous phage. Thus, a downstreamtranslatable DNA sequence encodes an amino acid residue sequence thatcorresponds, and preferably is identical, to the filamentous phage geneIII or gene IX coat polypeptide. The sequences of such coat proteins areknown and accessible in public databases such as the NCBI.

A cDNA or genomic library can be screened using a probe based upon thesequence of a polynucleotide of the present invention, such as thosedisclosed herein. Probes can be used to hybridize with genomic DNA orcDNA sequences to isolate homologous genes in the same or differentorganisms. Those of skill in the art will appreciate that variousdegrees of stringency of hybridization can be employed in the assay; andeither the hybridization or the wash medium can be stringent. As theconditions for hybridization become more stringent, there must be agreater degree of complementarity between the probe and the target forduplex formation to occur. The degree of stringency can be controlled byone or more of temperature, ionic strength, pH and the presence of apartially denaturing solvent, such as formamide. For example, thestringency of hybridization is conveniently varied by changing thepolarity of the reactant solution through, for example, manipulation ofthe concentration of formamide within the range of 0% to 50%. The degreeof complementarity (sequence identity) required for detectable bindingwill vary in accordance with the stringency of the hybridization mediumand/or wash medium. The degree of complementarity will optimally be100%, or 70-100%, or any range or value therein. However, it should beunderstood that minor sequence variations in the probes and primers canbe compensated for by reducing the stringency of the hybridizationand/or wash medium.

In one aspect of the invention, the polynucleotides are constructedusing techniques for incorporation of randomized codons in order tovariegate the resulting polypeptide at one or more specific residues orto add residues at specific locations within the sequence. Variousstrategies may be used to create libraries of altered polypeptidesequences including random, semi-rational and rational methods. Rationaland semi-rational methods have the advantage over the random strategiesin that one has more control over the consequences of changes introducedinto the coding sequence. In addition, by focusing the variation incertain regions of the gene, the universe of all possible amino acidvariants can be explored in chosen positions.

A library built on the common NNK or NNS diversification schemeintroduce a possible 32 different codons in every position and all 20amino acids. Such a library theoretically grows by 32n for every nnumber of residues. In practical terms, however, phage display islimited to sampling libraries of 10⁹ to 10¹⁰ variants implying that only6-7 residues can be targeted for variegation if full sequence coverageis to be achieved in the library. Thus, semi-rational or “focused”methods to generate libraries of scaffold variants by identifying keypositions to be variegated and choosing the diversification regimeaccording can be applied. A “codon set” refers to a set of differentnucleotide triplet sequences used to encode desired variant amino acids.A standard form of codon designation is that of the IUB code, which isknown in the art and described herein. A “non-random codon set” refersto a codon set that encodes select amino acids. Synthesis ofoligonucleotides with selected nucleotide “degeneracy” at certainpositions is well known in that art, for example the TRIM approach(Knappek et al.; J. Mol. Biol. (1999), 296:57-86); Garrard & Henner,Gene (1993), 128:103). Such sets of nucleotides having certain codonsets can be synthesized using commercially available nucleotide ornucleoside reagents and apparatus.

A codon set is a set of different nucleotide triplet sequences used toencode desired variant amino acids. Codon sets can be represented usingsymbols to designate particular nucleotides or equimolar mixtures ofnucleotides as shown in below according to the IUB code.

IUB Codes

G Guanine

A Adenine

T Thymine

C Cytosine

R (A or G)

Y (C or T)

M (A or C)

K (G or T)

S(C or G)

W (A or T)

H (A or C or T)

B (C or G or T)

V (A or C or G)

D (A or G or T)

N (A or C or G or T)

For example, in the codon set DVK, D can be nucleotides A or G or T; Vcan be A or G or C; and K can be G or T. This codon set can present 18different codons and can encode amino acids Ala, Tip, Tyr, Lys, Thr,Asn, Lys, Ser, Arg, Asp, Glu, Gly, and Cys.

Focused (e.g., non-random) libraries can be generated using NNK codonsand focusing the varigation at selected residues or, alternatively,variants with non-random substitutions can be generated using forexample DVK codons, which encodes 11 amino acids (ACDEGKNRSYW) and onestop codon. Alternatively, Kunkel mutagenesis can be used to variegatethe desired residues or regions of the polypeptide (Kunkel et al.,Methods Enzymol. 154:367-382, 1987).

Standard cloning techniques are used to clone the libraries into avector for expression. The library may be expressed using known system,for example expressing the library as fusion proteins. The fusionproteins can be displayed on the surface of any suitable phage. Methodsfor displaying fusion polypeptides comprising antibody fragments on thesurface of a bacteriophage are well known (U.S. Pat. No. 6,969,108 toGriffith; U.S. Pat. No. 6,172,197 to McCafferty; U.S. Pat. No. 5,223,409to Ladner; U.S. Pat. No. 6,582,915 to Griffiths; U.S. Pat. No. 6,472,147to Janda). Libraries for de novo polypeptide isolation can be displayedon pIX (WO2009085462A1). The libraries can also be translated in vitro,for example using ribosome display (Hanes and Pluckthun, Proc. Natl.Acad. Scie. USA, 94:4937, 1997), mRNA display (Roberts and Szostak,Proc. Natl. Acad. Sci. USA, 94:12297, 1997), CIS-display (Odegrip et.al., Proc. Natl. Acad. Sci. USA, 101:2806, 2004) or other cell-freesystems (U.S. Pat. No. 5,643,768 to Kawasaki).

Libraries with diversified regions can be generated using vectorscomprising the polynucleotide encoding the Tencon sequence (SEQ ID NO:16) or a predetermined mutant thereof. The template construct may have apromoter and signal sequences for the polypeptide chain. To makescaffold libraries, mutagenesis reactions using oligonucleotides thatcoded for loop regions (A:B, B:C, C:D, D:E, E:F, and F:G) of thescaffold are used. To ensure the incorporation of all chosen positionsinto the randomization scheme, a stop codon (such as TAA) can beincorporated in each region desired to be intended to be diversified.Only clones where the stop codons have been replaced will occur.

Modified Scaffold Polypeptides

Modified protein scaffolds and fragments of the invention can compriseone or more moieties that are covalently bonded, directly or indirectly,to another protein.

In the case of the addition of peptide residues, or the creation of anin-line fusion protein, the addition of such residues may be throughrecombinant techniques from a polynucleotide sequence as describedherein. In the case of an appended, attached or conjugated peptide,protein, organic chemical, inorganic chemical or atom, or anycombination thereof, the additional moiety that is bonded to a proteinscaffold or fragment of the invention is typically via other than apeptide bond. The modified protein scaffolds of the invention can beproduced by reacting a protein scaffold or fragment with a modifyingagent. For example, the organic moieties can be bonded to the proteinscaffold in a non-site specific manner by employing an amine-reactivemodifying agent, for example, an NHS ester of PEG. Modified proteinscaffolds and fragments comprising an organic moiety that is bonded tospecific sites of a protein scaffold of the present invention can beprepared using suitable methods, such as reverse proteolysis (Fisch etal., Bioconjugate Chem., 3:147-153 (1992); Werlen et al., BioconjugateChem., 5:411-417 (1994); Kumaran et al., Protein Sci. 6(10):2233-2241(1997); Itoh et al., Bioorg. Chem., 24(1): 59-68 (1996); Capellas etal., Biotechnot Bioeng., 56(4):456-463 (1997)), and the methodsdescribed in Hermanson, G. T., Bioconjugate Techniques, Academic Press:San Diego, Calif. (1996).

Where a polymer or chain is attached to the scaffold protein, thepolymer or chain can independently be a hydrophilic polymeric group, afatty acid group or a fatty acid ester group. As used herein, the term“fatty acid” encompasses mono-carboxylic acids and di-carboxylic acids.A “hydrophilic polymeric group,” as the term is used herein, refers toan organic polymer that is more soluble in water than in octane. Forexample, polylysine is more soluble in water than in octane. Thus, aprotein scaffold modified by the covalent attachment of polylysine isencompassed by the invention. Hydrophilic polymers suitable formodifying protein scaffolds of the invention can be linear or branchedand include, for example, polyalkane glycols (e.g., PEG,monomethoxy-polyethylene glycol (mPEG), PPG and the like), carbohydrates(e.g., dextran, cellulose, oligosaccharides, polysaccharides and thelike), polymers of hydrophilic amino acids (e.g., polylysine,polyarginine, polyaspartate and the like), polyalkane oxides (e.g.,polyethylene oxide, polypropylene oxide and the like) and polyvinylpyrolidone. Preferably, the hydrophilic polymer that modifies theprotein scaffold of the invention has a molecular weight of about 800 toabout 150,000 Daltons as a separate molecular entity. For example,PEG₅₀₀₀ and PEG_(20,000), wherein the subscript is the average molecularweight of the polymer in Daltons, can be used. The hydrophilic polymericgroup can be substituted with one to about six alkyl, fatty acid orfatty acid ester groups. Hydrophilic polymers that are substituted witha fatty acid or fatty acid ester group can be prepared by employingsuitable methods. For example, a polymer comprising an amine group canbe coupled to a carboxylate of the fatty acid or fatty acid ester, andan activated carboxylate (e.g., activated with N,N-carbonyl diimidazole)on a fatty acid or fatty acid ester can be coupled to a hydroxyl groupon a polymer.

Fatty acids and fatty acid esters suitable for modifying proteinscaffolds of the invention can be saturated or can contain one or moreunits of unsaturation. Fatty acids that are suitable for modifyingprotein scaffolds of the invention include, for example, n-dodecanoate(C₁₂, laurate), n-tetradecanoate (C₁₄, myristate), n-octadecanoate (C₁₈,stearate), n-eicosanoate (C₂₀, arachidate), n-docosanoate (C₂₂,behenate), n-triacontanoate (C₃₀), n-tetracontanoate (C₄₀),cis-Δ9-octadecanoate (C₁₈, oleate), all cis-Δ5,8,11,14-eicosatetraenoate(C₂₀, arachidonate), octanedioic acid, tetradecanedioic acid,octadecanedioic acid, docosanedioic acid, and the like. Suitable fattyacid esters include mono-esters of dicarboxylic acids that comprise alinear or branched lower alkyl group. The lower alkyl group can comprisefrom one to about twelve, preferably, one to about six, carbon atoms.

Fc-containing proteins can be compared for functionality by severalwell-known in vitro assays. In particular, affinity for members of theFcγRI, FcγRII, and FcγRIII family of Fcγ receptors is of interest. Thesemeasurements could be made using recombinant soluble forms of thereceptors or cell-associated forms of the receptors. In addition,affinity for FcRn, the receptor responsible for the prolongedcirculating half-life of IgGs, can be measured, for example, by BIAcoreusing recombinant soluble FcRn. Cell-based functional assays, such asADCC assays and CDC assays, provide insights into the likely functionalconsequences of particular variant structures. In one embodiment, theADCC assay is configured to have NK cells be the primary effector cell,thereby reflecting the functional effects on the FcγRIIIA receptor.Phagocytosis assays may also be used to compare immune effectorfunctions of different variants, as can assays that measure cellularresponses, such as superoxide or inflammatory mediator release. In vivomodels can be used as well, as, for example, in the case of usingvariants of anti-CD3 antibodies to measure T cell activation in mice, anactivity that is dependent on Fc domains engaging specific ligands, suchas Fcγ receptors.

Host Cell Selection or Host Cell Engineering

As described herein, the host cell chosen for expression of thescaffold-based protein is an important contributor to the finalcomposition, including, without limitation, the variation in compositionof the oligosaccharide moieties decorating the protein, if desirable,for example in the immunoglobulin CH2 domain when present. Thus, oneaspect of the invention involves the selection of appropriate host cellsfor use and/or development of a production cell expressing the desiredtherapeutic protein.

Further, the host cell may be of mammalian origin or may be selectedfrom COS-1, COS-7, HEK293, BHK21, CHO, BSC-1, Hep G2, 653, SP2/0, 293,HeLa, myeloma, lymphoma, yeast, insect or plant cells, or anyderivative, immortalized or transformed cell thereof.

Alternatively, the host cell may be selected from a species or organismincapable of glycosylating polypeptides, e.g. a prokaryotic cell ororganism, such as and of the natural or engineered E. coli spp,Klebsiella spp., or Pseudomonas spp.

Selecting Binding Domains

The polypeptides or fusion proteins or components and domains thereofmay also be obtained from selecting from libraries of such domains orcomponents, e.g., a phage library. A phage library can be created byinserting a library of random oligonucleotides or a library ofpolynucleotides containing sequences of interest, such as antibodydomains from the B-cells of an immunized animal or human (Smith, G. P.1985. Science 228: 1315-1317). Antibody phage libraries contain heavy(H) and light (L) chain variable region pairs in one phage allowing theexpression of single-chain Fv fragments or Fab fragments (Hoogenboom, etal. 2000, Immunol. Today 21(8) 371-8). The diversity of a phagemidlibrary can be manipulated to increase and/or alter the specificities ofthe polypeptides of the library to produce and subsequently identifyadditional, desirable, molecular properties and the polynucleotidesencoding them.

Other libraries of target binding components which may include otherthan antibody variable regions are ribosome display, CIS-display, yeastdisplay, bacterial displays and mammalian cell display. Ribosome displayis a method of translating mRNAs into their cognate proteins whilekeeping the protein attached to the RNA. The nucleic acid codingsequence is recovered by RT-PCR (Mattheakis, L. C. et al. 1994. Proc.Natl. Acad. Sci. USA 91, 9022). CIS-display is an alternative in vitrodisplay method in which the library is constructed as a fusion proteinwith RepA. During in vitro translation, RepA binds in cis to the DNAwhich it was made from, providing a direct linkage between genotype andphenotype (Odegrip et. al., Proc. Natl. Acad. Sci. USA, 101:2806, 2004).Yeast display is based on the construction of fusion proteins of themembrane-associated alpha-agglutinin yeast adhesion receptor, aga1 andaga2, a part of the mating type system (Broder, et al. 1997. NatureBiotechnology, 15:553-7). Bacterial display is based on fusion of thetarget to exported bacterial proteins that associate with the cellmembrane or cell wall (Chen and Georgiou 2002. Biotechnol Bioeng,79:496-503). Similarly, mammalian display systems are based on thecreation of a fusion protein between the polypeptide containingrandomized sequences and a secreted, membrane anchor protein.

Uses of Scaffold-Based Molecules

The compositions of the scaffold-based molecules described herein andgenerated by any of the above described methods may be used to diagnose,monitor, modulate, treat, alleviate, help prevent the incidence of, orreduce the symptoms of human disease or specific pathologies in cells,tissues, organs, fluid, or, generally, a host. A scaffold-based moleculeengineered for a specific purpose may be used to treat animmune-mediated or immune-deficiency disease, a metabolic disease, acardiovascular disorder or disease; a malignant disease; neurologicdisorder or disease; an infection such as a bacterial, viral orparasitic infection; or other known or specified related conditionincluding swelling, pain, and tissue necrosis or fibrosis.

Such a method can comprise administering an effective amount of acomposition or a pharmaceutical composition comprising at least onescaffold protein to a cell, tissue, organ, animal or patient in need ofsuch modulation, treatment, alleviation, prevention, or reduction insymptoms, effects or mechanisms. The effective amount can comprise anamount of about 0.001 to 500 mg/kg per single (e.g., bolus), multiple orcontinuous administration, or to achieve a serum concentration of0.01-5000 ug/ml serum concentration per single, multiple, or continuousadministration, or any effective range or value therein, as done anddetermined using known methods, as described herein or known in therelevant arts.

Compositions Comprising Scaffold-Based Proteins

The target binding scaffold proteins which are modified or unmodified,monovalent, bi- or multivalent, and mono-, bi- or multi-targeting, canbe isolated using separation procedures well known in the art forcapture, immobilization, partitioning, or sedimentation and purified tothe extent necessary for commercial applicability.

For therapeutic use, the scaffold-base proteins may be formulated of anappropriate mode of administration including but not limited toparenteral, subcutaneous, intramuscular, intravenous, intrarticular,intrabronchial, intraabdominal, intracapsular, intracartilaginous,intracavitary, intracelial, intracerebellar, intracerebroventricular,intracolic, intracervical, intragastric, intrahepatic, intramyocardial,intraosteal, intrapelvic, intrapericardiac, intraperitoneal,intrapleural, intraprostatic, intrapulmonary, intrarectal, intrarenal,intraretinal, intraspinal, intrasynovial, intrathoracic, intrauterine,intravesical, intralesional, bolus, vaginal, rectal, buccal, sublingual,intranasal, or transdermal means. At least one protein scaffoldcomposition can be prepared for use in the form of tablets or capsules;powders, nasal drops or aerosols; a gel, ointment, lotion, suspension orincorporated into a therapeutic bandage or “patch” delivery system asknown in the art. The invention provides for stable formulations of anscaffold-base proteins, which is preferably an aqueous phosphatebuffered saline or mixed salt solution, as well as preserved solutionsand formulations as well as multi-use preserved formulations suitablefor pharmaceutical or veterinary use, comprising at least onescaffold-base protein in a pharmaceutically acceptable formulation.Suitable vehicles and their formulation, inclusive of other humanproteins, e.g., human serum albumin, are described, for example, in e.g.Remington: The Science and Practice of Pharmacy, 21^(st) Edition, Troy,D. B. ed., Lipincott Williams and Wilkins, Philadelphia, Pa. 2006, Part5, Pharmaceutical Manufacturing pp 691-1092, See especially pp. 958-989.

The compositions may be used with, or incorporate within a singleformulation, other actives known to be beneficial for treatment of theindicated disorder, condition, or disease or may be a tested bypreparing combinations of scaffold-based proteins with novelcompositions and actives.

While having described the invention in general terms, the embodimentsof the invention will be further disclosed in the following examplesthat should not be construed as limiting the scope of the claims.

EXAMPLE 1 Construction of Fc Glycosylation Variants

Tencon Design

The third FN3 domain from human Tenascin (SEQ ID NO: 3) can be used asan alternative scaffold capable of being engineered to bind to specifictarget molecules via surface exposed loops structurally analogous toantibody complementarity determining regions (CDR). The meltingtemperature of this domain is 54° C. in PBS in its native form. In orderto produce a scaffold molecule with a similar structure and improvedphysical properties, such as an improved thermal stability, a consensussequence was designed based on an alignment of 15 FN3 domains from humanTenascin (SEQ ID NOS: 1-15).

Analysis of the multiple sequence alignment in Table 1 shows that these15 domains have sequence identities to each other ranging from 13 to80%, with an average sequence identity among pairs of 29%. A consensussequence (SEQ ID NO: 16) was designed by incorporating the mostconserved (frequent) amino acid at each position from the alignmentshown in Table 1. In pairwise alignments, the consensus sequence of thepresent invention (SEQ ID NO: 16), designated as Tencon, is identical tothe FN3 domains from Tenascin at 34-59% of positions with an averagesequence identity of 43%.

Protein Expression and Purification

The amino acid sequence of Tencon (SEQ ID NO: 16) was back translated,resulting in the DNA sequence shown in SEQ ID NO: 17. This sequence wasassembled by overlapping PCR, subcloned into a modified pET15 vector,transformed into BL21Star(DE3) E. coli (Invitrogen) and plated onto LBagar plates containing 75 μg/mL carbenicillin. A single colony waspicked and grown overnight at 37° C. in 50 ml of TB media containing 2%glucose and 100 μg/mL carbenicillin. This culture was used to seed 500mL of autoinduction media (Overnight Express Instant TB media, Novagen)in a 2.5 L Ultra Yield flask (Thomson Instrument Company). The growthand expression was done using a dual program (3 hours at 37° C., 300rpm, followed by 16 hours at 30° C., 250 rpm) in an ATR Multitronshaking incubator.

The culture was harvested and centrifuged at 7000 rpm for 15 minutes ina JL8.1 rotor to pellet the cells. The cells were resuspended in 30 mlbuffer containing 20 mM sodium phosphate, pH 7.5, 500 mM NaCl, 10%glycerol, 20 mM imidazole, 0.37 mg/mL lysozyme, 1× Complete Proteaseinhibitor (EDTA-free; Roche) and Benzonase (Sigma-Aldrich, 0.25 μl/mlfinal) and lysed with a Misonix XL2020 sonicator for 5 minutes on ice inpulse mode (5 seconds on, 30 seconds off). The insoluble material wasremoved by centrifugation at 17,000 rpm for 30 minutes in a JA-17 rotor.

The Tencon protein was purified from the soluble lysate in a 2-stepchromatographic process. First, the protein was captured by immobilizedmetal affinity chromatography, adding 2 mL Ni-NTA agarose beads (Qiagen)to the lysate and placing it on a rocking platform for 1 hour at 4° C.The resin was then packed into a Poly-Prep column (Bio-Rad) and washedwith 20 mM sodium phosphate, pH 7.5, 500 mM NaCl, 10% glycerol and 20 mMimidazole to remove the unbound material. The proteins were eluted fromthe resin with 20 mM sodium phosphate, pH 7.5, 500 mM NaCl, 10% glyceroland 500 mM imidazole. The fractions were analyzed by SDS-PAGE, both byCoomassie stain and by Western blot using an HRP-conjugated anti-Hisantibody (Immunology Consultants Laboratory). The desired fractions werepooled and dialyzed into PBS pH 7.4. As a second purification step theprotein was loaded onto a Superdex-75 HiLoad 16/60 column (GEHealthcare) equilibrated in PBS. The fractions were analyzed bySDS-PAGE, and the fractions containing Tencon were pooled andconcentrated using a Centriprep UltraCel YM-3 concentrator (Amicon).

Protein concentration was determined using a BioTek plate reader tomeasure the absorbance of the sample at 280 nm. The final preparationwas analyzed by Coomassie stain (FIG. 1), Western blot with anti-Hisantibody, and by HPLC-SEC using a G3000SW-XL column (TOSOH Biosciences)equilibrated in PBS. SDS-PAGE analysis shows that Tencon migratesbetween 6 and 14 kDa, in agreement with the expected mass of 10.7 kDafor the monomeric protein. A yield of >50 mg of pure Tencon protein perliter of culture was obtained.

Biophysical Characterization

The structure and stability of Tencon was characterized by circulardichroism spectroscopy and differential scanning calorimetryrespectively. CD measurements were made on an AVIV spectrometer at 20°C. in PBS and a concentration of 0.2 mg/mL. The spectrum in FIG. 8 showsa minimum at 218 nm, suggestive of beta-sheet structure as expected fora protein belonging to the FN3 family as designed. DSC data was obtainedby heating 0.5 mg/mL solutions of the 3^(rd) FN3 domain from Tenascin orTencon in PBS from 35° C. to 95° C. at a rate of 1° C./minute in anN-DSCII calorimeter (Applied Thermodynamics). First, the curve for thebuffer blank was subtracted to produce the profiles shown in FIG. 3.From this data, melting temperatures of 54° C. and 78° C. werecalculated for the 3^(rd) FN3 domain and Tencon, respectively, usingCpCalc (Applied Thermodynamics) software. The folding and unfolding ofboth domains is reversible at these temperatures.

Immunogenicity Analysis

A computer program that models for immunogenicity to human of amino acidsequences was used to compare the predicted immunogenicity of amino acidsequences representing the 3^(rd) FN3 domain of human Tenascin, Tencon,and several therapeutic antibodies (shown in Table 2). Chimeric mAbs anda human mAb (adalimumab) analyzed with the program were followed byapplication of a tolerance threshold (removes 9-mer peptides with 100%identity to human germline encoded sequence). The tolerance thresholdwas not applied to Tenascin or Tencon. The tolerance threshold assumesbroad T cell tolerance to germline encoded mAb sequences and focusesanalyses on novel sequence primarily in CDRs and flanking domains.

These analyses predict a low immunogenic risk for both Tenascin andTencon based on the likelihood that a 9-mer peptide, derived from theanalyzed sequence will bind one or more HLA molecules. The score isweighted with respect to the prevalence of each HLA allele. The scoresfor the models were summed for each sequence to provide a single numberdescribing the overall PIR of each sequence (score sum). The resultsfrom this analysis are summarized in Table 2. Tenascin was shown to havethe lowest overall Score (11.9). Tencon, like Tenascin, scored primarilynon-binders and low predicted immunogenic risk agretopes (Score=13.2).The Tenascin and Tencon sequences scored favorably as compared to thetherapeutic antibodies.

Display of Tencon on M13 Phage by pIX Fusion

The gene encoding the Tencon amino acid sequence was subcloned into thephagemid expression vector pPep9 by PCR and restriction digest cloning,resulting in the vector pTencon-pIX. This system expresses N-terminallyMyc-tagged Tencon as a C-terminal fusion to the N-terminus of the M13pIX protein (FIG. 4). The Lac promoter allows for lower levels ofexpression without IPTG and increased expression after the addition ofIPTG. The OmpA signal sequence was appended to the N-terminus of Tenconto promote efficient translocation to the periplasm. A short TSGGGGSlinker (SEQ ID NO: 141) was constructed between Tencon and pIX toprevent steric interactions between these proteins.

For confirmation of display on the surface of the M13 phage particle,pTencon-pIX was transformed into XL 1-Blue E. coli and a single colonywas used to innoculate a 5 mL LB culture supplemented with ampicillin.This culture was grown at 37° C. until reaching mid-log phase at whichpoint 6¹⁰ pfu of VCSM13 helper phage was added and the culture incubatedat 37° C. for 10 minutes without shaking followed by 50 minutes withshaking. The helper phage rescued culture was then diluted into 50 mL of2YT media supplemented with ampicillin and kanamycin and grown at 37° C.with shaking until O.D.₆₀₀ reached 0.7, at which point IPTG was added toa final concentration of 1 mM and the temperature reduced to 30° C.After 16 hours, the culture was centrifuged at 4000×g for 20 minutes andthe supernatant collected and stored at 4° C. for analysis.

Binding of the phage particles to an anti-Myc antibody (Invitrogen) wasused to confirm the display of the Myc-Tencon construct on the M13 phagesurface. A Maxisorp plate was coated overnight at a concentration of 2.5ug/mL with anti-Myc or an anti-av antibody (negative control) andblocked with SuperBlock T20 (Pierce). Two-fold serial dilutions of thephagemid culture supernatant described above were made in PBS and addedto the wells of the coated plate. After 1 hour, the plate was washedwith TBST and a anti-M13 HRP antibody was added to each well and washedwith TBST following a 1-hour incubation. The Roche BD ELISA PODsubstrate was added and luminescence detected on a plate reader (Tecan).FIG. 5 shows that the Myc-Tencon phage particles bind to the anti-myc,but not the anti-αv antibody coated wells or the uncoated control wellsof the plate in a concentration dependent manner, confirming thespecific display of Myc-Tencon on the M13 phage particle.

An additional phagemid vector can be constructed to display Tencon andlibrary members (see Example 2) on M13 phage as fusions to coat proteinpIII. For this system, the gene for pIX is replaced with a gene encodinga truncated version of pIII (Bass et al. 1990). Additional changes ascompared to the system shown in FIG. 4 include the replacement of theOmpA signal sequence with the signal sequence for DsbA, as secretionusing this sequence has been shown to be beneficial for the display ofstable alternative scaffold molecules (Steiner et al. 2006).

EXAMPLE 2 Generation of Tencon Libraries

Tencon variant libraries can be made by many different methods,depending on the desired complexity and the relative location ofmutations in the molecule. DNA synthesis methods are preferred to createmutations scattered throughout the Tencon gene. Restriction enzymecloning can also be used to recombine DNA fragments containing mutationsin different regions of the gene. Saturating mutagenesis in asmall-defined region, such as a single Tencon loop, can be introduced byusing a degenerate oligo-nucleotide and oligonucleotide directedmutagenesis (Kunkel et al. 1987).

A Tencon library, library FG7, designed to replace the FG loop with 7random amino acids using oligonucleotide directed mutagenesis wasconstructed. An oligonucleotide (TconFG7-For-5′pho) was synthesized tohave a 21 base pair (bp) degenerate sequence of NNS at the positionsencoding the FG loop and two flanking 20-27 bp nucleotide sequences ofcomplementarity to the Tencon coding sequence. In this design, alltwenty amino acids are capable of being represented in the FG loop. Thecalculated diversity at nucleotide level is 1.3×10⁹.

TconFG7-For5′pho: (SEQ ID NO: 18)GAATACACCGTTTCTATCTACGGTGTTNNSNNSNNSNNSNNSNNSNNSC CGCTGTCTGCGGAATTCAC

The template for oligonucleotide directed mutagenesis,pDsbA-Tencon-Asc-loop-Myc-pIII, was constructed by replacing the TenconF:G loop encoding sequence with a stem loop sequence containing an AscIrestriction site. This system allows the elimination of backgroundtemplate DNA after mutagenesis by digesting the resulting DNA with AscIprior to transformation. To purify a single-stranded DNA template formutagenesis, a single colony of E. coli CJ236 harboringpDsbA-Tencon-Asc-loop-Myc-pIII, was picked into 5 mL of 2YT growthmedium with carbenicillin (50 ug/ml final concentration) andChloramphenicol (10 ug/ml). After 6 hours, VCSM13 helper phage was addedto a final concentration of 10¹⁰ pfu/ml and incubated without shakingfor 10 minutes before being transferred to 150 mL of 2YT withcarbenicillin (10 ug/ml) and uridine (0.25 ug/ml) and incubated at 37°C. with shaking at 200 rpm overnight. The cells were pelleted bycentrifugation and the supernatant collected and the phage pelleted withPEG NaCl. Single strand DNA was purified from this pellet using aQIAprep Spin M13 kit (Qiagen) according to the manufacturerinstructions.

To anneal the degenerate oligonucleotide to the template, 5 ug oftemplate DNA was combined with oligo TconFG7-For-5-pho at a molar ratioof 10:1 in Tris-HCl (50 mM, pH7.5) and MgC12 (10 mM) and incubated at90° C. for 2 minutes, 60° C. for 3 minutes, and 20° C. for 5 minutes.After the annealing reaction, ATP (10 mM), dNTPs (25 mM each), DTT (100mM), T4 ligase (7 units), and T7 DNA polymerase (10 units) were added tothe reaction mixture and incubated at 14° C. for 6 hours followed by 20°C. for 12 hours. The resulting DNA was purified using a PCR purificationkit (Qiagen) and recovered in 100 uL of water. The library DNA wasdigested with 10 units of AscI for 4 hours and then purified again withQiagen PCR purification kit. The final library DNA was recovered in 50uL of water. The resulting double stranded DNA product was thentransformed into E. coli MC1061F′ by electroporation.

The transformants were collected in 20 mL SOC medium and allowed torecover for 1 hour at 37° C. At the end of the recovery, an aliquot ofthe transformation was serial diluted and plated on Carbenicillin (100ug/ml) plates containing 1% glucose to assess the total transformantnumber. The remaining SOC culture was then used to inoculate 1 L of 2xYTmedium with Carbinicillin and 1% glucose and grown until OD₆₀₀ reached0.6. 100 mL of this culture was inoculated with M13 helper phage to10¹⁰/mL and incubated at 37° C. before centrifugation. The resultingcell pellet was resuspended in 500 mL fresh 2xYT medium containingCarbenicillin (100 ug/mL) and Kanamycin (35 ug/mL) and grown at 30° C.overnight before centrifugation. Phage particles were precipitated bythe addition of PEG/NaCl and stored at −80° C.

A second library, BC6/FG7, was designed to introduce diversity withinthe B:C and F:G loops of Tencon simultaneously. In order to do so, twooligonucleotides, Tc-BC6-For-5′phos and POP149 were synthesized. Theforward oligo was phosphorylated and contained 18 bases of NNS codon ateach position encoding the B:C loop, while the reverse oligo wasbiotinylated at the 5′ end and contained 21 bases of NNS codon at eachposition encoding the F:G loop. Both oligonucleotides are flanked by two18 bp nucleotide sequences identical to the region preceding andfollowing the region to be mutagenized (see below for primer detail).

Tc-BC6-For-5′phos: (SEQ ID NO: 19)gactctctgcgtctgtctggNNSNNSNNSNNSNNSNNSTTCGACTCTTT CCTGATCCAGTACCPOP 2149: (SEQ ID NO: 20)GTGAATTCCGCAGACAGCGGSNNSNNSNNSNNSNNSNNSNNAACACCGT AGATAGAAACGGTG

To construct the library, sixteen 100 uL PCR reactions were performedusing t oligos Tc-CB6-For5′phos and POP2149 to amplify the Tencon DNAtemplate, introducing NNS codons into the B:C and F:G loopssimultaneously in the process. The double-stranded PCR product was mixedwith magnetic streptavidin beads (Dynal) in B&W buffer (10 mM Tris-HCl,pH7.5, 1 mM EDTA, 2M NaCl, 0.1% Tween-20) and incubated for 20 minutes,pulled down with a magnet and washed with B&W buffer twice. The forwardstrand was eluted from the beads with 300 uL of 150 mM NaOH. This“megaprimer,” a mixture of long primers with more than 8×10¹⁶ intheoretical diversity, was used to anneal to a single strand librarytemplate. Library construction was carried out as described above forthe FG7 library.

EXAMPLE 3 Selection of IgG Binders

In order to perform selections of Tencon library members that bind toIgG, recombinant IgG (human IgG1 subtype) was biotinylated usingsulfo-NHS-LC-Biotin (Pierce) before dialyzing into PBS. For selections,200 uL of phage displaying libraries FG7 or BC6/FG7 were blocked with200 uL of chemiblocker before the addition of biotinylated IgG atconcentrations of 500 nM (round 1) or 100 nM (rounds 2 and 3). Boundphages were recovered by Neutravidin magnetic beads (Seradyne) in round1 or streptavidin magnetic beads (Promega) in rounds 2 and 3. Unboundphages were washed from the beads using 5-10 washes with 1 mL of Trisbuffered saline with tween (TBST) followed by 21 mL washes with Trisbuffered saline (TBS). Bound phages were eluted from the beads by theaddition of mid-log phase E. coli MC1061F′. Infected cells were platedon LB agar plates supplemented with carbenicillin and glucose. The nextday, cells were scraped from the plate and grown to mid-log phase beforerescue with VCSM13 helper phage and grown overnight. Phage particlesisolated by PEG/NaCl precipitation and used for the next round ofselections.

After 3 rounds of panning against IgG, the output was subcloned into apET27 vector modified to include a ligase independent cloning site byamplifying the Tencon gene by PCR. This PCR product was annealed to thevector and transformed into BL21-GOLD(DE3) cells (Stratagene).Individual colonies were picked into 1 mL cultures in 96 deep wellplates (Corning) and grown to saturation overnight at 37° C. The nextday, 50 microL of the overnight culture was used to inoculate a fresh 1mL culture. Cultures were grown at 37° C. for 2 hours before adding IPTGto 1 mM and reducing the temperature to 30° C. Cells were harvested bycentrifugation 16 hours after induction and lysed with 100 microL ofBugBuster (Novagen). The resulting lysates were clarified bycentriguation and used to test for binding to IgG by ELISA.

Maxisorp plates (Nunc) were coated with 0.1 μg of anti-HIS antibody(Qiagen) overnight, washed with TBST, and blocked with Starting BlockT20 (Thermo Scientific). Clarified lysates diluted 1:4 in Starting Blockwere added to the plates and allowed to bind for 1 hour before washingwith TBST. Biotinylated IgG or biotinylated HSA was added at aconcentration of 1 ug/ml and washed with TBST after a 1-hour incubation.Detection of bound IgG or HSA was accomplished by addingstreptavidin-HRP (Jackson Immunoresearch) and detecting with PODchemiluminescence substrate. Results of the ELISA are shown in FIG. 7.Constructs that bound biotinylated IgG more than 10-fold overbiotinylated HSA as judged by ELISA signal were sequenced. Aftercompletion of several selection experiments, 60 unique binding sequencesfrom library FG7 and 10 unique sequences from library BC6FG7 wereobtained; Table 4 shows representative sequences of IgGbinders in whichthe B:C and/or F:G loops are shown to the extent they are different thanthose of SEQ ID NO:16. Also shown Table 4 are numerous mutations inother regions of the scaffold.

The Tencon protein designed, expressed, and purified here has a thermalstability improved by 26° C. with respect to that of the 3^(rd) FN3domain from human Tenascin, which has been used as an alternativescaffold molecule. Based on this stability increase, this scaffoldmolecule is likely to be more amenable to amino acid substitution andeasier to manufacture. Mutations that decrease protein stability arelikely to be better tolerated in the context of a more stable scaffoldand thus a scaffold with enhanced stability is likely to yield morefunctional, well folded binders from a library of scaffold variants.

TABLE 1  SEQ. ID. NOS:(1)1        |10        |20       |30       |40        |50       |60 1(1)---SPPKDLVVTEVTEETVNLAWDN-EMRVTEYLVVYTPTH--EGGLEMQFRVPGDQTSTI 2(1)TYLPAPEGLKFKSIKETSVEVEWDPLDIAFETWEIIFRNMN-KEDEGEITKSLRRPETSYR 3(1)---DAPSQIEVKDVTDTTALITFIFKPLAEIDGIELTYGIKD--VPGDRTTIDLTEDENQS 4(1)TGLDAPRNLRRVSQTDNSITLEWRNGKAAIDSYRIKYAPISGGDHAEVDVPKSQQATTKTT 5(1)---DTPKDLQVSETAETSLTLLWKTPLAKFDRYRLNYSLPT----GQWVGVQLPRNTTSYV 6(1)-QAPELENLTVTEVGWDGLRLNWTAADQAYEHFIIQVQEAN--KVEAARNLTVPGSLRAVD 7(1)-ETPNLGEVVVAEVGWDALKLNWTAPEGAYEYFFIQVQEAD--TVEAAQNLTVPGGLRSTD 8(1)-EVPDMGNLTVTEVSWDALRLNWTTPDGTYDQFTIQVQEAD--QVEEAHNLTVPGSLRSME 9(1)-DLPQLGDLAVSEVGWDGLRLNWTAADNAYEHFVIQVQEVN--KVEAAQNLTLPGSLRAVD 10(1)-KEPEIGNLNVSDITPESFNLSWMATDGIFETFTIEIIDSN--RLLETVEYNISGAERTAH 11(1)-ALPLLENLTISDINPYGFTVSWMASENAFDSFLVTVVDSG--KLLDPQEFTLSGTQRKLE 12(1)-AEPEVDNLLVSDATPDGFRLSWTADEGVFDNFVLKIRDTK--KQSEPLEITLLAPERTRD 13(1)---GSPKEVIFSDITENSATVSWRAPTAQVESFRITYVPITG---GTPSMVTVDGTKTQTR 14(1)---DGPSGLVTANITDSEALARWQPAIATVDSYVISYTGEK----VPEITRTVSGNTVEYA 15(1)---DSPRDLTATEVQSETALLTWRPPRASVTGYLLVYESVD----GTVKEVIVGPDTTSYS                       SEQ.          ID. NOS:        |70       |80       |90     100 1IQELEPGVEYFIRVFAILENKKSIPVSARVAT------- 2QTGLAPGQEYEISLHIVKNNTRGPGLKRVTTTRLD---- 3IGNLKPDTEYEVSLISRRGDMSSNPAKETFTT------- 4LTGLRPGTEYGIGVSAVKEDKESNPATINAATELDTPKD 5LRGLEPGQEYNVLLTAEKGRHKSKPAKSKPARVK----- 6IPGLKAATPYTVSIYGVIQGYRTPVLSAEASTGE----- 7LPGLKAATHYTITIRGVTQDFSTTPLSVEVLTE------ 8IPGLRAGTPYTVTLHGEVRGHSTRPLAVEVVTE------ 9IPGLEAATPYRVSIYGVIRGYRTPVLSAEASTAKEPE-- 10ISGLPPSTDFIVYLSGLAPSIRTKTISATATTE------ 11LRGLITGIGYEVMVSGFTQGHQTKPLRAEIVTE------ 12LTGLREATEYEIELYGISKGRRSQTVSAIATTAM----- 13LVKLIPGVEYLVSIIAMKGFEESEPVSGSFTTAL----- 14LTDLEPATEYTLRIFAEKGPQKSSTITAKFTTDL----- 15LADLSPSTHYTAKIQALNGPLRSNMIQTIFTTIGL----

TABLE 2 1st 2^(nd) Score Score Score Score sum sum Sequence Descriptionsum sum (chain) (molecule) Tenascin Alt. Scaff. 6.01 5.85 11.86 11.86Tencon Alt. Scaff. 5.83 7.37 13.20 13.20 adalimumab Vh humanized mAb9.45 8.06 17.50 45.42 Vl 15.29 12.63 27.92 cetuximab Vh Chimeric mAb17.63 16.89 34.52 64.44 Vl 14.45 15.47 29.92 Rituximab Vh Chimeric mAb16.57 14.38 30.96 61.65 Vl 16.63 14.06 30.69 basiliximab Vh Chimeric mAb16.48 13.40 29.89 58.98 Vl 16.05 13.05 29.09

TABLE 3 Loops Loop Residues of SEQ ID NO: 16 Amino Acid Sequence A-B13-16 TEDS (SEQ ID NO: 16) B-C 22-28 TAPDAAF (SEQ ID NO: 16) C-D 38-43SEKVGE (SEQ ID NO: 16) D-E 51-54 GSER (SEQ ID NO: 16) E-F 60-64 GLKPG(SEQ ID NO: 16) F-G 75-81 KGGHRSN (SEQ ID NO: 16)

TABLE 4 Scaffolds binding to IgG B:C Loop F:G Loop Residues 22-28Residues 75-81 Clone No. (SEQ ID NO) (SEQ ID NO) Scaffold Mutations 1SYGFNN (21) QIGPIIP (46) 2 TYEGES (22) QIGPIIP (46) 3 TYESES (23)QIGPIIP (46) 4 TNWMDS (24) SIRTIDS (47) 5 KSVFIM (25) PKFHSPL (48) 6YSSYAT (26) WKTTIWF (49) 7 RFHPFP (27) RKNWKTR (50) 8 MMCMPL (28)RLFRIYQ (51) 9 YCRVRD (29) WLSRSYD (52) 10 SYGFNN (21) WLSRSYD (52) 11MDCFMG (30) WLSRSCD (53) 12 TYRFNS (31) WMGPYCD (54) 13 ASRRSL (32)RRRRYSF (55) 14 TIESES (33) HIVPMVP (56) 15 TL*MQS (34) QIEPIIR (57) 16IYDSES (35) PSAANNP (58) 17 VRLRYVQ (59) 18 QVGPLIP (60) 19 RIGPILP (61)20 QIGPLLP (62) 21 RIGPLLP (63) 22 QVGPLLP (64) 23 RIGPMLP (65) 24QIGPVLP (66) 25 RIGPVLP (67) 26 QIGPMMP (68) 27 QVGPLVP (69) 28 QIGPMLP(70) R18P 29 QVGPILP (71) 30 QVGPLLP (64) 31 QVGPMLP (72) 32 QIGPIVP(73) I33V 33 MIGPLLP (74) 34 QIGPLFP (75) 35 QIGPVLP (66) T59A 36QIGPMVP (76) 37 QIGPIVP (77) 38 RIEPILP (78) V74G 39 VAGSVWP (79) 40REGATLY (80) 41 KQIPPIL (81) S38G 42 LSLSSVL (82) 43 HMLLPLP (83) V74A44 MIGPLIP (84) 45 TIGPHIP (85) 46 EIGPCLP (86) 47 EIGPVLP (87) 48KIGPCLP (88) Y35H 49 MIGPVLP (89) 50 QIGPILP (90) S52P 51 QIGPILP (90)Q36R 52 QIGPILP (90) 53 EVGPILP (91) 54 QVGPLLP (92) A23T 55 QIGPVMP(93) 56 QIGPCVP (94) 57 QIGPLVP (95) 58 RGLVMPM (96) V74A 59 MIGPILP(97) 60 QIGPILP (90) E37G 61 QIGPILP (90) T68A 62 QIGPILP (90) T22I 63QIGPILP (90) S52F 64 QIGPILP (90) Y56H 65 QIGPILP (90) A44V 66 QIGPILP(90) P24S 67 RIGPILP (61) 68 CIGPMVP (98) 69 FIGPVLP (99) 70 HIGPILP(100) 71 HIGPIMP (101) 72 HIGPYLP (102) 73 HVGPILP (103) 74 IIGPLLP(104) 75 LIGPLLP (105) 76 MVGPLLP (106) 77 NIGPYLP (107) 78 NIGPYLP(108) 79 QIGPHLP (109) 80 QIGPIIP (46) 82 QIGPILG (110) 83 QIGPILS (111)83 QIGPILT (112) 84 QIGPIMP (113) 85 QIGPIPI (114) 86 QIGPLLN (115) 87QIGPLLP (62) 88 QIGPVFP (116) 89 QIGPVLS (117) 90 QIGPWLP (118) 92QVGPILP (71) 93 QVGPILR (118) 94 QVGPIMN (119) 95 QVGPIMP (120) 96QVGPIVP (121) 97 QVGPLLS (122) 98 QVGPVLP (123) 99 QVGPVLT (124) 100RIGPIMP (125) 101 RIGPIVP (126) 102 RIGPMFP (127) 103 RIGPMIP (128) 104RIGPMVP (129) 105 RIGPVIP (130) 106 RVGPILP (131) 107 RVGPLLP (132) 108TVGPHIP (133) 109 DRKRFI (36) PSWRSNW (134) 110 EFWRGS (37) QIGPLLP (62)111 GLLDPL (38) ALRATLE (135) 112 GLVLPE (39) KYGYLTP (136) 113 MASDGL(40) RIGPMLP (137) 114 NKTETN (41) NPFCSRF (138) 115 QAERKV (42) QIGPLLP(62) 116 QAERKV (42) RIGPLLP (63) 117 SQVCTL (43) YYLHQWC (139) 118YFDKDS (44) QIGPLLP (62) 119 YFECEP (45) HIVPLLR (140)Sequences:

SEQ ID No. 1: sppkdlvvtevteetvnlawdnemrvteylvvytpthegglemgfrvpgdgtstiiqelepgveyfirvfailenkksipvsarvat SEQ ID No. 2:tylpapeglkfksiketsvevewdpldiafetweiifrnmnkedegeitkslrrpetsyrqtglapgqeyeislhivknntrgpglkrvtttrld SEQ ID No. 3:dapsqievkdvtdttalitwfkplaeidgieltygikdvpgdrttidltedenqysignlkpdteyevslisrrgdmssnpaketftt SEQ ID No. 4tgldaprnlrrvsqtdnsitlewrngkaaidsyrikyapisggdhaevdvpksqqattkttltglrpgteygigvsavkedkesnpatinaateldtp kd SEQ ID No. 5dtpkdlqvsetaetsltllwktplakfdryrlnyslptgqwvgvqlprnttsyvlrglepgqeynvlltaekgrhkskpakskparvk SEQ ID No. 6qapelenltvtevgwdglrlnwtaadqayehfiiqvqeankveaarnltvpgslravdipglkaatpytvsiygviqgyrtpvlsaeastge SEQ ID No. 7etpnlgevvvaevgwdalklnwtapegayeyffiqvqeadtveaaqnltvpgglrstdlpglkaathytitirgvtqdfsttplsvevlte SEQ ID No. 8evpdmgnltvtevswdalrlnwttpdgtydqftiqvqeadqveeahnltvpgslrsmeipglragtpytvtlhgevrghstrplavevvte SEQ ID No. 9dlpqlgdlavsevgwdglrlnwtaadnayehfviqvqevnkveaaqnltlpgslravdipgleaatpyrvsiygvirgyrtpvlsaeastakepe SEQ ID No. 10kepeignlnvsditpesfnlswmatdgifetftieiidsnrlletveynisgaertahisglppstdfivylsglapsirtktisatatte SEQ ID No. 11alpllenltisdinpygftvswmasenafdsflvtvvdsgklldpqeftlsgtqrklelrglitgigyevmvsgftqghqtkplraeivte SEQ ID No. 12aepevdnllvsdatpdgfrlswtadegvfdnfvlkirdtkkqsepleitllapertrdltglreateyeielygiskgrrsqtvsaiattam SEQ ID No. 13gspkevifsditensatvswraptaqvesfrityvpitggtpsmvtvdgtktqtrlvklipgveylvsiiamkgfeesepvsgsfttal SEQ ID No. 14dgpsglvtanitdsealarwqpaiatvdsyvisytgekvpeitrtvsgntveyaltdlepateytlrifaekgpqksstitakfttdl SEQ ID No. 15dsprdltatevqsetalltwrpprasvtgyllvyesvdgtvkevivgpdttsysladlspsthytakiqalngplrsnmiqtifttigl SEQ ID No. 16LPAPKNLVVSEVTEDSLRLSWTAPDAAFDSFLIQYQESEKVGEAINLTVPGSERSYDLTGLKPGTEYTVSIYGVKGGHRSNPLSAEFTT SEQ ID No. 17ctgccggcgccgaaaaacctggttgtttctgaagttaccgaagactctctgcgtctgtcttggaccgcgccggacgcggcgttcgactctttcctgatccagtaccaggaatctgaaaaagttggtgaagcgatcaacctgaccgttccgggttctgaacgttcttacgacctgaccggtctgaaaccgggtaccgaatacaccgtttctatctacggtgttaaaggtggtcaccgttctaaccc gctgtctgcggaattcaccaccTencon Sequence Showing Loops (SEQ ID NO: 16)

              A-B       B-C             C-D1-LPAPKNLVVSEVTEDSLRLSWTAPDAAFDSFLIQYQESEKVGEA-44         D-E       E-F            F-G 45-INLTVPGSERSYDLTGLKPGTEYTVSIYGVKGGHRSNPLSAEFTT-89

EXAMPLE 4 Stabilizing Mutations of Tencon

Mutants were designed to improve the folding stability of Tenconscaffold described herein above (SEQ ID NO: 16). Several point mutationswere made to produce substitution of individual residues of SEQ ID NO:16, such as N46V (Tencon17—SEQ ID NO:142), E14P (Tencon18—SEQ IDNO:143), E11N (Tencon19—SEQ ID NO:144), E37P (Tencon20—SEQ ID NO:145),and G73Y (Tencon21—SEQ ID NO:146) which were predicted to improvestability by the program PoPMuSiC v2.0 (Dehouck, Grosfils et al. 2009).The mutant E86I (Tencon22—SEQ ID NO:147) had been previously found tostabilize a homologous protein, the 3^(rd) FN3 domain from humanTenascin (W02009/086116A2). Finally, the L17A mutation was found tosignificantly stabilize Tencon during alanine scanning experiments inwhich all loop residues of Tencon were replaced with alanineindependently (data not shown). Following an initial round of stabilityassays (see below), the combinatorial mutants N46V/E86I (Tencon 23—SEQID NO:148), E14P/N46V/E86I (Tencon24—SEQ ID NO:149), and L17A/N46V/E86I(Tencon25—SEQ ID NO:150) were produced to further increase stability.

Expression and Purification

Mutations in the Tencon coding sequence were made using a QuikChangemutagenesis kit (Stratagene). The resulting plasmids were transformedinto BL21-GOLD (DE3) E. coli (Stratagene) for expression. A singlecolony was picked and grown overnight at 37° C. in 2 mL of TB mediacontaining 100 μg/ml ampicillin. This culture was used to seed 100 mL ofautoinduction media (Overnight Express Instant TB media, Novagen) in a500 mL baffled flask and grown at 37° C. for 16 hours.

The culture was harvested by centrifugation at 4000×g for 20 min and thepelleted cells resuspended 5 mL of BugBuster HT (Novagen) per gram ofwet cell pellet. After 30 minutes of incubation at room temperature,lysates were clarified by centrifugation at 30,000×g for 20 minutes andloaded onto a 3 mL Ni-NTA superflow column (Novagen) by gravity. Afterloading, each column was washed with 15 mL of a buffer containing 50 mMsodium phosphate pH 7.4, 500 mM NaCl, and 10 mM imidazole. Bound proteinwas then eluted from the column using 10 mL of a buffer containing 50 mMsodium phosphate pH 7.4, 500 mM NaCl, and 250 mM imidazole. Proteinpurity was assessed by SDS-PAGE. Prior to biophysical analysis, eachmutant was dialyzed thoroughly into PBS pH 7.4. 28-33 mg of purifiedprotein was obtained for each mutant from 100 mL of culture.

Characterization of Thermal Stability

The thermal stabilities of the parent Tencon and each mutant weremeasured by capillary differential scanning calorimetry (DSC). Eachsample was dialyzed extensively against PBS pH 7.4 and diluted to aconcentration of 2-3 mg/mL. Melting temperatures were measured for thesesamples using a VP-DSC instrument equipped with an autosampler(MicroCal, LLC). Samples were heated from 10° C. to 95° C. or 100° C. ata rate of 1° C. per minute. A buffer only scan was completed betweeneach sample scan in order to calculate a baseline for integration. Datawere fit to a two state unfolding model following subtraction of thebuffer only signal. Reversibility of thermal denaturation was determinedby repeating the scan for each sample without removing it from the cell.Reversibility was calculated by comparing the area under the curve fromthe 1^(st) scan with the 2^(nd) scan. Results of the DSC experiments arepresented in Table 5 as the values derived from complete melting curves.Single mutants Tencon17, Tencon18, Tencon19, and Tencon22 improved thethermal stability compared to the parent tencon sequence. Only Tencon21was significantly destabilizing. Combinatorial mutant samples Tencon23,Tencon24, and Tencon25 all had a significantly larger enhancement of thestability, indicating that the designed mutations are additive withrespect to improving thermal stability.

Denaturation by Guandine Hydrochloride

The abilities of Tencon and each mutant to remain folded upon treatmentwith increasing concentrations of guanidine hydrochloride (GdmCl) asmeasured by tryptophan fluorescence were used to assess stability.Tencon contains only one tryptophan residue. The tryptophan residue isburied within the hydrophobic core and thus fluorescence emission at 360nm is a sensitive measure of the folded state of this protein. 200 uL ofa solution containing 50 mM sodium phosphate pH 7.0, 150 mM NaCl, andvariable concentrations of GdmCl from 0.48 to 6.63 M were pipetted intoblack, non-binding, 96-well plates (Greiner) in order to produce a 17point titration. 10 uL of a solution containing the tencon mutants wereadded to each well across the plate to make a final proteinconcentration of 23 uM and mixed by pipetting up and down gently. Afterincubation at room temperature for 24 hours, fluorescence was read usinga Spectramax M5 plate reader (Molecular Devices) with excitation at 280nm and emission at 360 nm. The data generated from such curves is shownin FIG. 8. Fluorescence signal was converted to fraction unfolded usingthe equation (Pace 1986 Methods Enzymol 131: 266-80):f _(u)=(γ_(F)−γ)/(γ_(F)−γ_(U))Where y_(F) is the fluorescence signal of the folded sample and y_(u) ofthe unfolded sample.The mid-points of the unfolding transition and slope of the transitionwere determined by fitting to the equation below (Clarke, Hamill et al.1997):

$F = \frac{\left( {\alpha_{N} + {\beta_{N}\lbrack D\rbrack}} \right) + {\left( {\alpha_{D} + {\beta_{D}\lbrack D\rbrack}} \right){\exp\left( {{m\left( {\lbrack D\rbrack - \lbrack D\rbrack_{50\%}} \right)}/{RT}} \right)}}}{1 + {\exp\left( {{m\left( {\lbrack D\rbrack - \lbrack D\rbrack_{50\%}} \right)}/{RT}} \right)}}$Where F is the fluorescence at the given denaturant concentration, α_(N)and α_(D) are the y-intercepts of the native and denatured state, β_(N)and β_(D) are the slopes of the baselines for the native and denaturedstate, [D] is the concentration of GdmCl, [D]_(50%) the GdmClconcentration at which point 50% of the sample is denatured, m the slopeof the transition, R the gas constant, and T the temperature. The freeenergy of folding for each sample was estimated using the equation (Pace1986 supra; Clarke, Hamill et al. 1997 J Mol Biol 270(5): 771-8):ΔG=m[D]_(50%)

It is often difficult to accurately measure the slope of the transition,m, for such curves. Additionally, the mutations described here are notexpected to alter the folding mechanism of tencon. Thus, the m value foreach mutant was measured and the values averaged (Pace 1986 supra) toproduce an m=3544 cal/mol/M used for all free energy calculations. Theresults of these calculations are presented in Table 5. The results forGdmCl unfolding experiments demonstrate that the same mutants thatstabilize Tencon with respect to thermal stability also stabilize theprotein against GdmCl induced denaturation.

Size Exclusion Chromatography

Size exclusion chromatography (SEC) was used to assess the aggregationstate of WT tencon and each mutant. 5 uL of each sample were injectedonto a Superdex 75 5/150 column (GE Healthcare) at a flow rate of 0.3mL/min with a PBS mobile phase. Elution from the column was monitored byabsorbance at 280 nm. In order to assess the aggregation state, thecolumn was previously calibrated with globular molecular weightstandards (SIGMA®). All of the samples tested, with the exception ofTencon21, eluted in one peak at an elution volume consistent with thatof a monomeric sample. Tencon21 eluted with 2 peaks, indicating thepresence of aggregates.

TABLE 5 Tm [D]_(50%) ΔG(H₂O) Construct Mutations (Kcal) (M) (kcal/mol)Tencon 16 78.04 3.4 12.0 (SEQ ID NO: 16) Tencon17 N46V 81.88 3.6 12.8(SEQ ID NO: 142) Tencon18 E14P 82.77 3.5 12.4 (SEQ ID NO: 143) Tencon19E11N 79.00 3.4 12.0 (SEQ ID NO: 144) Tencon20 E37P 77.40 3.4 12.0 (SEQID NO: 145) Tencon21 G73Y 67.56 2.4 8.5 (SEQ ID NO: 146) Tencon22 E86I82.78 3.7 13.1 (SEQ ID NO: 147) Tencon23 N46V/E86I 86.65 4.1 14.5 (SEQID NO: 148) Tencon24 E14P/N46V/E86I 87.47 4.0 14.2 (SEQ ID NO: 149)Tencon25 L17A/N46V/E86I 92.73 5.1 18.1 (SEQ ID NO: 150) Tencon26 L17A84.9 4.6 16.2 (SEQ ID NO: 151)

It will be clear that the invention can be practiced otherwise than asparticularly described in the foregoing description and examples.Numerous modifications and variations of the present invention arepossible in light of the above teachings and, therefore, are within thescope of the appended claims.

What is claimed:
 1. A method of constructing a library of ascaffold-based protein derived from a stability enhanced consensussequence of a fibronectin type III domain incorporating randomizedcodons in order to produce polypeptide variants, comprising the stepsof: providing a polynucleotide sequence encoding a polypeptidecomprising a stability enhanced consensus sequence of a fibronectin typeIII domain selected from the group consisting of SEQ ID NOs: 142-151;introducing randomizing codons into the polynucleotide sequence atselected positions; and propagating copies of the polynucleotidesequence to form a library of polynucleotides encoding variant scaffoldproteins.
 2. The method of claim 1, wherein the randomizing codons areselected from the group NNS and NNK.
 3. The method of claim 2, whereinthe randomizing codons are incorporated at positions encoding at leastone loop region selected from the group consisting of residues at orabout positions 13-16, 22-28, 38-43, 51-54, 60-64, and 75-81 of SEQ IDNO: 142-151.
 4. A library produced by the method of claim 3.